*/Disease Information (sort of like the Intro to your Mini Research Write up): Staphylococcus aureus is a gram positive bacterium commonly found among the human body. It is a common cause of several infections, and is increasingly growing resistant to antibiotics. Methicillin resistant Staphylococcus aureus (MRSA) is the leading cause of nosocomial, or hospital-acquired, pneumonia and is the second leading cause of nosocomial blood stream infections. MRSA is becoming more prevalent within the United States, and as a result, an increasing demand for novel antibiotics has emerged.
Link to TDR Targets page (if present): not available
Essentiality of this protein:
D-alanine ligase is an essential enzyme in bacterial cell wall biosynthesis and an important target
for developing new antibiotics. Mutations in the protein has caused resistance against the antibiotic that's used
as last resort, D-Alanine Metabolism plays a role in the metabolism of other amino acids. D-Alanine ligase is an
essential enzyme in bacterial cell wall biosynthesis.
Figure 1. Reaction catalyzed by D-alanine: D-alanine ligase. (EC 6.3.2.4)
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): Malachite green phosphate assay
-- link to Sigma (or other company) page for assay (see Sigma links below) -- -or link (or citation) to paper that contains assay information https://www.caymanchem.com/pdfs/10009325.pdf
Image of protein (PyMol with features delineated and shown separately):
Figure 2. Crystal structure of D-alanine: D-alanine Ligase from Staphylococcus aureus,shown asPyMOL
representation.Protein chains shown as cartoon and sticks colored separately on spectrum gradient. (PDB: 3N8D)
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): mtkenicivfggksaehevsiltaqnvlnaidkdkyhvdiiyitndgdwrkqnnitaeik stdelhlengealeisqllkesssgqpydavfpllhgpngedgtiqglfevldvpyvgng vlsaassmdklvmkqlfehrglpqlpyisflrseyekyehnilklvndklnypvfvkpan lgssvgiskcnneaelkegikeafqfdrklvieqgvnareievavlgndypeatwpgevv kdvafydykskykdgkvqlqipadldedvqltlrnmaleafkatdcsglvradffvtedn qiyinetnampgftafsmypklwenmglsypelitklielakerhqdkqknkykid
*length of your protein in Amino Acids: 356
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website 40 kDa (40230.5 Da)
Molar Extinction coefficient of your protein at 280 nm wavelength: 40465 M^-1 cm^-1 (assuming all pairs of Cys residues form cystines) 40340 M^-1 cm^-1 (assuming all Cys residues are reduced)
Prefix: SaDAL
*NCBI Gene # or RefSeq#: 23197880
*Protein ID (NP or XP #) or Wolbachia#: KEK52203.1
*Organism (including strain): Staphylococcus aureus
Etiologic Risk Group (see link below):
*/Disease Information (sort of like the Intro to your Mini Research Write up):
Staphylococcus aureus is a gram positive bacterium commonly found among the human body. It is a common cause of several
infections, and is increasingly growing resistant to antibiotics. Methicillin resistant Staphylococcus aureus (MRSA) is the leading
cause of nosocomial, or hospital-acquired, pneumonia and is the second leading cause of nosocomial blood stream infections.
MRSA is becoming more prevalent within the United States, and as a result, an increasing demand for novel antibiotics has
emerged.
Link to TDR Targets page (if present): not available
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
NCBI -
http://www.ncbi.nlm.nih.gov/gene/?term=23197880
UniProt -
http://www.uniprot.org/uniprot/Q2FWH3#entry_information
PATRIC -
https://www.patricbrc.org/portal/portal/patric/Feature?cType=feature&cId=PATRIC.985762.7.JPRT010
00013.CDS.45731.46801.fwd
Essentiality of this protein:
D-alanine ligase is an essential enzyme in bacterial cell wall biosynthesis and an important target
for developing new antibiotics. Mutations in the protein has caused resistance against the antibiotic that's used
as last resort, D-Alanine Metabolism plays a role in the metabolism of other amino acids. D-Alanine ligase is an
essential enzyme in bacterial cell wall biosynthesis.
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html):
Most likely a 2-mer (dimer)
Complex of proteins?: dimer
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
*EC#: 6.3.2.4
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/enzyme.php?ecno=6.3.2.4
Figure 1. Reaction catalyzed by D-alanine: D-alanine ligase. (EC 6.3.2.4)
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): Malachite green phosphate assay
-- link to Sigma (or other company) page for assay (see Sigma links below)
-- -or link (or citation) to paper that contains assay information
https://www.caymanchem.com/pdfs/10009325.pdf
-- links to assay reagents (substrates) pages.
--- List cost and quantity of substrate reagents, supplier, and catalog no.
AquaPhoenix ATP (2g), Fisher-Scientific, Catalog No. S25123…………………..$54.00
https://www.fishersci.com/shop/products/aquaphoenix-adenosine-triphosphate-atp/s25123
D-Alanine, +99% (5g), Fisher-Scientific, Catalog No. AC172080050…………….$30.00
https://www.fishersci.com/shop/products/d-alanine-99-acros-organics-3/p-4259097
Structure (PDB):
4 structures available: 3N8D, 2I80, 2I8C, 2I87
Structures for Screening: 3N8D (Anthony) ???? (Kevin)
Current Inhibitors:
vancomycin (developed resistance); Bruton's tyrosine kinase inhibitor LFM-A13
Expression Information (has it been expressed in bacterial cells):
Successfully expressed in E. coli M15 (pREP4)
http://www.pnas.org/content/103/41/15178
Purification Method: Nickel affinity chromatography
Image of protein (PyMol with features delineated and shown separately):
Figure 2. Crystal structure of D-alanine: D-alanine Ligase from Staphylococcus aureus,shown asPyMOL
representation.Protein chains shown as cartoon and sticks colored separately on spectrum gradient. (PDB: 3N8D)
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
mtkenicivfggksaehevsiltaqnvlnaidkdkyhvdiiyitndgdwrkqnnitaeik stdelhlengealeisqllkesssgqpydavfpllhgpngedgtiqglfevldvpyvgng vlsaassmdklvmkqlfehrglpqlpyisflrseyekyehnilklvndklnypvfvkpan lgssvgiskcnneaelkegikeafqfdrklvieqgvnareievavlgndypeatwpgevv kdvafydykskykdgkvqlqipadldedvqltlrnmaleafkatdcsglvradffvtedn qiyinetnampgftafsmypklwenmglsypelitklielakerhqdkqknkykid
*length of your protein in Amino Acids: 356
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website
40 kDa (40230.5 Da)
Molar Extinction coefficient of your protein at 280 nm wavelength:
40465 M^-1 cm^-1 (assuming all pairs of Cys residues form cystines)
40340 M^-1 cm^-1 (assuming all Cys residues are reduced)
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
ATGACAAAAGAAAATATTTGTATCGTTTTTGGAGGGAAAAGTGCAGAACACGAAGTATCG ATTCTGACAGCACAAAATGTATTAAATGCAATAGATAAAGACAAATATCATGTTGATATC ATTTATATTACCAATGATGGTGATTGGAGAAAGCAAAATAATATTACAGCTGAAATTAAA TCTACTGATGAGCTTCATTTAGAAAATGGAGAGGCGCTTGAGATTTCACAGCTATTGAAA GAAAGTAGTTCAGGACAACCATACGATGCAGTATTCCCATTATTACATGGTCCTAATGGT GAAGATGGCACGATTCAAGGGCTTTTTGAAGTTTTGGATGTACCATATGTAGGAAATGGT GTATTGTCAGCTGCAAGTTCTATGGACAAACTTGTAATGAAACAATTATTTGAACATCGA GGGTTACCACAGTTACCTTATATTAGTTTCTTACGTTCTGAATATGAAAAATATGAACAT AACATTTTAAAATTAGTAAATGATAAATTAAATTACCCAGTCTTTGTTAAACCTGCTAAC TTAGGGTCAAGTGTAGGTATCAGTAAATGTAATAATGAAGCGGAACTTAAAGAAGGTATT AAAGAAGCATTCCAATTTGACCGTAAGCTTGTTATAGAACAAGGCGTTAACGCACGTGAA ATTGAAGTAGCAGTTTTAGGAAATGACTATCCTGAAGCGACATGGCCAGGTGAAGTCGTA AAAGATGTCGCGTTTTACGATTACAAATCAAAATATAAAGATGGTAAGGTTCAATTACAA ATTCCAGCTGACTTAGACGAAGATGTTCAATTAACGCTTAGAAATATGGCATTAGAGGCA TTCAAAGCGACAGATTGTTCTGGTTTAGTCCGTGCTGATTTCTTTGTAACAGAAGACAAC CAAATATATATTAATGAAACAAATGCAATGCCTGGATTTACGGCTTTCAGTATGTATCCA AAGTTATGGGAAAATATGGGCTTATCTTATCCAGAATTGATTACAAAACTTATCGAGCTT
GCTAAAGAACGTCACCAGGATAAACAGAAAAATAAATACAAAATTGACTAA
*GC% Content for gene: 33.8
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
ATGACCAAAGAAAATATCTGCATCGTTTTCGGTGGTAAGTCTGCGGAACACGAAGTTTCT ATTCTGACCGCGCAGAATGTTCTCAACGCGATCGACAAAGATAAATACCACGTTGACATC ATCTATATTACCAATGACGGTGACTGGCGTAAACAAAACAATATCACGGCGGAAATCAAG AGCACGGACGAGCTGCATCTGGAAAATGGTGAGGCGCTGGAAATCTCTCAGCTCCTGAAA GAATCTTCTAGCGGTCAACCGTACGACGCAGTTTTTCCGCTGCTGCATGGCCCGAATGGC GAGGATGGTACTATCCAAGGTCTGTTCGAAGTTCTGGACGTTCCTTATGTTGGTAACGGT GTGCTGTCTGCAGCGTCTTCTATGGATAAACTCGTCATGAAGCAGCTGTTTGAACACCGT GGTCTGCCGCAGCTCCCGTACATCTCTTTCCTGCGTTCTGAATATGAAAAATACGAACAC AACATCCTGAAGCTGGTGAACGACAAACTGAACTATCCTGTTTTTGTTAAGCCAGCGAAT CTGGGCAGCTCTGTTGGCATCTCTAAATGCAACAACGAAGCCGAACTCAAAGAAGGTATC AAGGAAGCGTTCCAGTTTGACCGTAAACTGGTAATTGAACAGGGTGTTAATGCGCGCGAG ATCGAGGTGGCTGTCCTGGGCAATGATTACCCAGAAGCAACCTGGCCGGGTGAAGTAGTT AAAGACGTCGCGTTTTATGACTATAAGTCTAAATACAAGGACGGTAAAGTTCAACTCCAG ATCCCAGCGGACCTGGACGAAGATGTTCAGCTCACCCTGCGCAACATGGCACTGGAAGCC TTCAAAGCGACCGACTGCTCTGGTCTGGTTCGTGCGGACTTTTTCGTCACCGAGGACAAC CAGATTTACATCAATGAGACCAATGCGATGCCGGGTTTCACTGCATTTTCTATGTACCCG AAACTGTGGGAGAATATGGGTCTGAGCTATCCGGAACTCATCACTAAACTGATCGAACTG
GCGAAAGAGCGTCACCAGGATAAGCAGAAGAATAAGTACAAAATTGACTAA
*GC% Content for gene (codon optimized): 47.6